This two-part project studies the organization and function of specialized membranes in neurons and glia. The first part aims to characterize calcium regulation during synaptic activity in parallel fiber- Purkinje cell synapses of the cerebellar cortex and in synapses of CA3 hippocampal pyramidal cells. New frozen sectioning techniques, in combination with scanning transmission electron microscopy (STEM), have permitted studies of structure and calcium regulation in directly frozen preparations of a new kind of organotypic culture of hippocampus. Structural analysis in conjunction with electrophysiology recordings has demonstrated excellent organization of the CA3 mossy fiber synapses in these cultures. We have also successfully obtained cryosections from these synapses, and preliminary elemental analysis has provided for the first time data on the concentrations and distributions of total calcium in a hippocampal cultures with intact circuitry. In part two, the expression, transport and assembly of specialized myelin membrane proteins are studied. Confocal light microscopy had previously shown that Schwann cells depend on microtubules for intracellular transport and assembly of myelin-specific proteins. We have now used microtubule assembly-disassembly experiments to show that the P0 protein, the myelin- associated glycoprotein (MAG), and laminin, which are targeted to discrete and different plasma membrane domains, are sorted into separate carrier vesicles as they exit the trans-Golgi network. Following sorting, certain carrier vesicles are transported along the myelin internode on microtubules, even though microtubules do not appear to selectively target these vesicles to specific membrane sites.